1-Desoxy-2-Methylene-19-Nor-Vitamin D Analogs and Their Uses

ABSTRACT

This invention discloses 1-desoxy-2-methylene-19-nor-vitamin D analogs, and specifically (20S)-25-hydroxy-1-desoxy-2-methylene-19-nor-vitamin D 3  and pharmaceutical uses therefor. This compound exhibits relatively high binding activity and pronounced activity in arresting the proliferation of undifferentiated cells and inducing their differentiation to the monocyte thus evidencing use as an anti-cancer agent especially for the treatment or prevention of leukemia, colon cancer, breast cancer, skin cancer or prostate cancer.

BACKGROUND OF THE INVENTION

This invention relates to vitamin D compounds, and more particularly to1-Desoxy-2-Methylene-19-Nor-Vitamin D analogs and their pharmaceuticaluses, and especially (20S)-25-hydroxy-1-desoxy-2-methylene-19-norvitaminD₃, its biological activities, and its pharmaceutical uses.

The natural hormone, 1α,25-dihydroxyvitamin D₃ and its analog in theergosterol series, i.e. 1α,25-dihydroxyvitamin D₂ are known to be highlypotent regulators of calcium homeostasis in animals and humans, andtheir activity in cellular differentiation has also been established,Ostrem et al., Proc. Natl. Acad. Sci. USA, 84, 2610 (1987). Manystructural analogs of these metabolites have been prepared and tested,including 1α-hydroxyvitamin D₃, 1α-hydroxyvitamin D₂, various side chainhomologated vitamins and fluorinated analogs. Some of these compoundsexhibit an interesting separation of activities in cell differentiationand calcium regulation. This difference in activity may be useful in thetreatment of a variety of diseases such as renal osteodystrophy, vitaminD-resistant rickets, osteoporosis, psoriasis, and certain malignancies.

Another class of vitamin D analogs, i.e. the so called 19-nor-vitamin Dcompounds, is characterized by the replacement of the A-ring exocyclicmethylene group (carbon 19), typical of the vitamin D system, by twohydrogen atoms. Biological testing of such 19-nor-analogs (e.g.,1α,25-dihydroxy-19-nor-vitamin D₃) revealed a selective activity profilewith high potency in inducing cellular differentiation, and very lowcalcium mobilizing activity. Thus, these compounds are potentiallyuseful as therapeutic agents for the treatment of malignancies, or thetreatment of various skin disorders. Two different methods of synthesisof such 19-nor-vitamin D analogs have been described (Perlman et al.,Tetrahedron Lett. 31, 1823 (1990); Perlman et al., Tetrahedron Lett. 32,7663 (1991), and DeLuca et al., U.S. Pat. No. 5,086,191).

In U.S. Pat. No. 4,666,634, 2β-hydroxy and alkoxy (e.g., ED-71) analogsof 1α,25-dihydroxyvitamin D₃ have been described and examined by Chugaigroup as potential drugs for osteoporosis and as antitumor agents. Seealso Okano et al., Biochem. Biophys. Res. Commun. 163, 1444 (1989).Other 2-substituted (with hydroxyalkyl, e.g., ED-120, and fluoroalkylgroups) A-ring analogs of 1α,25-dihydroxyvitamin D₃ have also beenprepared and tested (Miyamoto et al., Chem. Pharm. Bull. 41, 1111(1993); Nishii et al., Osteoporosis Int. Suppl. 1, 190 (1993); Posner etal., J. Org. Chem. 59, 7855 (1994), and J. Org. Chem. 60, 4617 (1995)).

2-substituted analogs of 1α,25-dihydroxy-19-nor-vitamin D₃ have alsobeen synthesized, i.e. compounds substituted at 2-position with hydroxyor alkoxy groups (DeLuca et al., U.S. Pat. No. 5,536,713), with 2-alkylgroups (DeLuca et at U.S. Pat. No. 5,945,410), and with 2-alkylidenegroups (DeLuca et al U.S. Pat. No. 5,843,928), which exhibit interestingand selective activity profiles. All these studies indicate that bindingsites in vitamin D receptors can accommodate different substituents atC-2 in the synthesized vitamin D analogs.

In a continuing effort to explore the 19-nor class of pharmacologicallyimportant vitamin D compounds, analogs which are characterized by thepresence of a methylene substituent at carbon 2 (C-2), a hydroxyl groupat carbon 1 (C-1), and a shortened side chain attached to carbon 20(C-20) have also been synthesized and tested.1α-hydroxy-2-methylene-19-nor-pregnacalciferol is described in U.S. Pat.No. 6,566,352 while 1α-hydroxy-2-methylene-19-nor-homopregnacalciferolis described in U.S. Pat. No. 6,579,861 and1α-hydroxy-2-methylene-19-nor-bishomopregnacalciferol is described inU.S. Pat. No. 6,627,622. All three of these compounds have relativelyhigh binding activity to vitamin D receptors and relatively high celldifferentiation activity, but little if any calcemic activity ascompared to 1α,25-dihydroxyvitamin D₃. Their biological activities makethese compounds excellent candidates for a variety of pharmaceuticaluses, as set forth in the '352, '861 and '622 patents.

Analogs of the natural hormone 1α,25-dihydroxyvitamin D₃ characterizedby the transposition of the A-ring exocyclic methylene group from carbon10 (C-10) to carbon 2 (C-2) (e.g.,1α,25-dihydroxy-2-methylene-19-norvitamin D analogs) have beensynthesized and tested [see Sicinski et al., J. Med. Chem., 41, 4662(1998); Sicinski et al., Steroids 67, 247 (2002); and, DeLuca et al.,U.S. Pat. Nos. 5,843,928; 5,936,133 and 6,382,071)]. Molecular mechanicsstudies performed on these analogs predict that a change of A-ringconformation may cause flattening of the cyclohexanediol ring. Molecularmechanics calculations and NMR studies also predict that the A-ringconformational equilibrium would be ca. 6:4 in favor of the conformerhaving an equatorial 1α-OH. It was further predicted that introductionof the 2-methylene group into 19-nor-vitamin D carbon skeleton wouldchange the character of its 1α- and 3β-A-ring hydroxyls. They would bothbe in allylic positions similar to the 1α-hydroxyl group in the moleculeof the natural hormone [i.e., 1α,25-(OH)₂D₃]. It was found that1α,25-dihydroxy-2-methylene-19-norvitamin D analogs are characterized bysignificant biological potency. In addition, the biological potency ofsuch analogs may be enhanced dramatically where “unnatural”(20S)-configuration is present. Taking into account these findings, thepresent invention is aimed at vitamin D compounds characterized by thetransposition of the A-ring exocyclic methylene group from carbon 10(C-10) to carbon 2 (C-2) (e.g., 2-methylene-19-norvitamin D analogs).Although these analogs lack 1α-OH, that is important for biologicalactivity, such hydroxyl group can be potentially introducedenzymatically in the living organisms.

SUMMARY OF THE INVENTION

The present invention is directed toward1-desoxy-2-methylene-19-nor-vitamin D analogs, and their pharmaceuticaluses, and more specifically toward(20S)-25-hydroxy-1-desoxy-2-methylene-19-norvitamin D₃, its biologicalactivity, and various pharmaceutical uses for this compound.

Structurally these 1-desoxy-2-methylene-19-nor-vitamin D analogs arecharacterized by the general formula I shown below:

where X is selected from the group consisting of hydrogen and ahydroxy-protecting group, and where the group R represents any of thetypical side chains known for vitamin D type compounds. Thus, R may bean alkyl, hydrogen, hydroxyalkyl or fluoroalkyl group, or R mayrepresent a side chain of the formula:

where Z in the above side chain structure is selected from Y, —OY,—CH₂OY, —C≡CY and —CH═CHY, where the double bond in the side chain mayhave the cis or trans geometry, and where Y is selected from hydrogen,methyl, —COR⁵ and a radical of the structure:

where m and n, independently, represent the integers from 0 to 5, whereR¹ is selected from hydrogen, deuterium, hydroxy, protected hydroxy,fluoro, trifluoromethyl, and C₁₋₅-alkyl, which may be straight chain orbranched and, optionally, bear a hydroxy or protected-hydroxysubstituent, and where each of R⁵, R³, and R⁴, independently, isselected from deuterium, deuteroalkyl, hydrogen, fluoro, trifluoromethyland C₁₋₅ alkyl, which may be straight-chain or branched, and optionally,bear a hydroxy or protected-hydroxy substituent, and where R¹ and R²,taken together, represent an oxo group, or an alkylidene group having ageneral formula C_(k)H_(2k)— where k is an integer, the group ═CR²R³, orthe group —(CH₂)_(p)—, where p is an integer from 2 to 5, and where R³and R⁴, taken together, represent an oxo group, or the group—(CH₂)_(q)—, where q is an integer from 2 to 5, and where R⁵ representshydrogen, hydroxy, protected hydroxy, or C₁₋₅ alkyl and wherein any ofthe CH-groups at positions 20, 22, or 23 in the side chain may bereplaced by a nitrogen atom, or where any of the groups —CH(CH₃)—,—(CH₂)_(m)—, —CR₁R₂— or —(CH₂)_(n)— at positions 20, 22, and 23,respectively, may be replaced by an oxygen or sulfur atom.

Specific important examples of side chains with natural20R-configuration are the structures represented by formulas (a), (b),(c), (d) and (e) below, i.e., the side chain as it occurs in25-hydroxyvitamin D₃ (a); vitamin D₃ (b); 25-hydroxyvitamin D₂ (C);vitamin D₂ (d); and the C-24 epimer of 25-hydroxyvitamin D₂ (e).

The wavy line to the carbon 20 indicates that carbon 20 may have eitherthe R or S configuration.

The preferred analog is(20S)-25-hydroxy-1-desoxy-2-methylene-19-norvitamin D₃ which has thefollowing formula Ia:

The above compounds of formula I, especially formula Ia, exhibit adesired, and highly advantageous, pattern of biological activity. Thesecompounds are characterized by relatively high binding to vitamin Dreceptors, i.e. they bind with only slightly lower affinity than1α,25-dihydroxyvitamin D_(3.) They are only slightly less potent causingdifferentiation of HL-60 cells than 1,25(OH)₂D₃. They also exhibitrelatively low transcriptional activity as well as relatively lowactivity in their ability to mobilize calcium from bone, and in theirability to promote intestinal calcium transport, as compared to1α,25-dihydroxyvitamin D₃. Hence, these compounds can be characterizedas having relatively little calcemic activity.

The above compounds I, and particularly Ia, have relatively high bindingaffinity, are characterized by relatively high cell differentiationactivity, but have notably lower calcemic activities. Thus, thesecompounds have potential as anti-cancer agents and provide therapeuticagents for the prevention or treatment of leukemia, colon cancer, breastcancer, skin cancer and prostate cancer.

One or more of the compounds may be present in a composition to treat orprevent the above-noted diseases in an amount from about 0.01 μg/gm toabout 1000 μg/gm of the composition, preferably from about 0.1 μg/gm toabout 500 μg/gm of the composition, and may be administered topically,transdermally, orally, rectally, nasally, sublingually, or parenterallyin dosages of from about 0.01 μg/day to about 1000 μg/day, preferablyfrom about 0.1 μg/day to about 500 μg/day.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-5 illustrate various biological activities of(20S)-25-hydroxy-1-desoxy-2-methylene-19-norvitamin D₃, hereinafterreferred to as “1-desoxy-2MD,” as compared to the native hormone1α,25-dihydroxyvitamin D₃, hereinafter “1,25(OH)₂D₃.”

FIG. 1 is a graph illustrating the relative activity of 1-desoxy-2MD and1,25(OH)₂D₃ to compete for binding with [³H]-1,25-(OH)₂-D₃ to thefull-length recombinant rat vitamin D receptor;

FIG. 2 is a graph illustrating the percent HL-60 cell differentiation asa function of the concentration of 1-desoxy-2MD and 1,25(OH)₂D₃;

FIG. 3 is a graph illustrating the in vitro transcription activity of1,25(OH)₂D₃ as compared to 1-desoxy-2MD;

FIGS. 4A and 4B are bar graphs illustrating the bone calciummobilization activity of 1,25(OH)₂D₃ as compared to 1-desoxy-2MD; and

FIGS. 5A and 5B are bar graphs illustrating the intestinal calciumtransport activity of 1,25(OH)₂D₃ as compared to 1-desoxy-2MD.

DETAILED DESCRIPTION OF THE INVENTION

As used in the description and in the claims, the term“hydroxy-protecting group” signifies any group commonly used for thetemporary protection of hydroxy functions, such as for example,alkoxycarbonyl, acyl, alkylsilyl or alkylarylsilyl groups (hereinafterreferred to simply as “silyl” groups), and alkoxyalkyl groups.Alkoxycarbonyl protecting groups are alkyl-O—CO— groupings such asmethoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl,butoxycarbonyl, isobutoxycarbonyl, tert-butoxycarbonyl,benzyloxycarbonyl or allyloxycarbonyl. The term “acyl” signifies analkanoyl group of 1 to 6 carbons, in all of its isomeric forms, or acarboxyalkanoyl group of 1 to 6 carbons, such as an oxalyl, malonyl,succinyl, glutaryl group, or an aromatic acyl group such as benzoyl, ora halo, nitro or alkyl substituted benzoyl group. The word “alkyl” asused in the description or the claims, denotes a straight-chain orbranched alkyl radical of 1 to 10 carbons, in all its isomeric forms.“Alkoxy” refers to any alkyl radical which is attached by oxygen, i.e. agroup represented by “alkyl-O—.” Alkoxyalkyl protecting groups aregroupings such as methoxymethyl, ethoxymethyl, methoxyethoxymethyl, ortetrahydrofuranyl and tetrahydropyranyl. Preferred silyl-protectinggroups are trimethylsilyl, triethylsilyl, t-butyldimethylsilyl,dibutylmethylsilyl, diphenylmethylsilyl, phenyldimethylsilyl,diphenyl-t-butylsilyl and analogous alkylated silyl radicals. The term“aryl” specifies a phenyl-, or an alkyl-, nitro- or halo-substitutedphenyl group.

A “protected hydroxy” group is a hydroxy group derivatised or protectedby any of the above groups commonly used for the temporary or permanentprotection of hydroxy functions, e.g. the silyl, alkoxyalkyl, acyl oralkoxycarbonyl groups, as previously defined. The terms “hydroxyalkyl”,“deuteroalkyl” and “fluoroalkyl” refer to an alkyl radical substitutedby one or more hydroxy, deuterium or fluoro groups respectively. An“alkylidene” refers to a radical having the general formula C_(k)H_(2k)—where k is an integer.

The preparation of 2-methylene-19-norvitamin D analogs of the basicstructure I can be accomplished by a common general method, i.e., thecondensation of a bicyclic Windaus-Grundmann type ketone II with theallylic phosphine oxide III:

In the structures II and III, groups X and R represent groups definedabove; X being preferably hydroxy-protecting group, it being alsounderstood that any functionalities in R that might be sensitive, orthat interfere with the condensation reaction, be suitable protected asis well-known in the art. The process shown above represents anapplication of the convergent synthesis concept, which has been appliedeffectively for the preparation of vitamin D compounds [e.g. Lythgoe etal., J. Chem. Soc. Perkin Trans. I, 590 (1978); Lythgoe, Chem. Soc. Rev.9, 449 (1983); Toh et al., J. Org. Chem. 48, 1414 (1983); Baggiolini etal., J. Org. Chem. 51, 3098 (1986); Sardina et al., J. Org. Chem. 51,1264 (1986); J. Org. Chem. 51, 1269 (1986); DeLuca et al., U.S. Pat. No.5,086,191; DeLuca et al., U.S. Pat. No. 5,536,713)].

Hydrindanones of the general structure II are known, or can be preparedby known methods. Specific important examples of such known bicyclicketones are the structures with the side chains (a), (b), (c) and (d)described above, i.e., 25-hydroxy Grundmann's ketone (e) [Baggiolini etal., J. Org. Chem, 51, 3098 (1986)]; Grundmann's ketone (f) [Inhoffen etal., Chem. Ber., 90, 664 (1957)]; 25-hydroxy Windaus ketone (g)[Baggiolini et al., J. Org. Chem., 51, 3098 (1986)] and Windaus ketone(h) [Windaus et al., Ann., 524, 297 (1936)]:

Regarding the preparation of the phosphine oxides of the structure III,alternative synthetic routes were established. As set forth in SCHEME I,an achiral, commercially available acetal-ketone 1, wasenantioselectively hydroxylated to the hydroxy ketone 2, using themethod elaborated by Hayashi et al. [J. Org. Chem. 69, 5966 (2004)] andinvolving the reaction of a ketone with nitrosobenzene in the presenceof a catalytic amount of L-proline. The introduced secondary hydroxylwas silylated and the protected compound 3 was subjected to the Wittigreaction with an ylide generated from methyltriphenylphosphonium bromideand n-butyllithium. In the resulting olefinic compound 4 the carbonylgroup was deprotected in the reaction with the Lewis acid (FeCl₃) andthe formed cyclohexanone 5 was subjected to a Peterson reaction leadingto the mixture of α,β-unsaturated esters 6 and 7. Although possible atthis stage, the separation of the geometric isomers was more easilyachieved (by column chromatography) after the reduction step, providingthe E- and Z-allylic alcohols 8 and 9, respectively. The E-isomer 8 wasnext transformed in the three-step procedure into the correspondingphosphine oxide 10. Wittig-Horner coupling of the known Grundmann ketone11 [see Sicinski et al., J. Med. Chem., 41, 4662 (1998)] with thelithium phosphinoxy carbanion generated from the phosphine oxide 10 wassubsequently carried out, producing the protected 19-norvitamin Dcompound, which after hydroxyls deprotection with tetrabutylammoniumfluoride provided the desired(20S)-2-methylene-25-hydroxy-19-nor-vitamin D₃ (12, 1-desoxy-2MD). Thissynthetic path is described in EXAMPLE I herein and the alternativemethod of the synthesis of the A-ring fragment, the phoshine oxide 27,and its coupling with the ketone 11 is described in EXAMPLE II herein.

SCHEME II shows this different synthetic sequence leading to thebuilding block 27 and to the final vitamin 12. As a chiral startingcompound served a commercially available D-(−)-quinic acid 13, which wasat first converted to the known lactone 14. Treatment of this compoundwith a 1,1′-thiocarbonyldiimidazole resulted in formation of the cyclicthiocarbonate 15 [see Mills et al., Tetrahedron Lett. 29, 281 (1988)].The Barton-McCombie deoxygenation reaction of the thiocarbonate 15 withtributyltin hydride and AIBN provided two isomeric products: the knowncompound 16 [see Gonzales-Bello et al., J. Chem. Soc., Perkin Trans. 1,849 (1999)] and the desired diol 17. Oxidation of the secondary hydroxylin the latter isomer yielded the ketone 18 which was subjected to theWittig methylenation. The lactone ring in the formed compound 19 wasthen opened and the secondary hydroxyl silylated. The methyl estermoiety in the obtained product 20 was reduced and the diol 21 wassubjected to periodate oxidation. Wittig reaction of the obtainedcyclohexanone 22 with methyl(triphenylphosphoranylidene)acetate providedthe mixture of α,β-unsaturated esters 23 and 24. They were reduced withDIBALH and the obtained allylic alcohols separated by columnchromatography. The E-isomer 25 was then converted into thecorresponding allylic phosphine oxide 27. Its anion, generated byphenyllitium, was coupled with the Grundmann ketone 11 and the final19-norvitamin 12 (1-desoxy-2MD) was obtained after acidic hydroxyldeprotection.

As it is evident from EXAMPLE I and II, other 19-norvitamin D analogshaving the different side-chains may be synthesized by the methods setforth herein.

This invention is described by the following illustrative examples. Inthese examples specific products identified by Arabic numerals (e.g., 1,2, 3, etc) refer to the specific structures so identified in thepreceding description and in the SCHEME I and SCHEME II.

EXAMPLES

Chemistry. Melting points (uncorrected) were determined on aThomas-Hoover capillary melting-point apparatus. Optical rotations weremeasured in chloroform using a Perkin-Elmer 241 automatic polarimeter at22° C. Ultraviolet (UV) absorption spectra were recorded with aPerkin-Elmer Lambda 3B UV-VIS spectrophotometer in ethanol. ¹H nuclearmagnetic resonance (NMR) spectra were recorded in deuteriochloroform at200, 400 and 500 MHz with a Varian Unity, Bruker DMX-400 and BrukerDMX-500 spectrometers, respectively. ¹³C nuclear magnetic resonance(NMR) spectra were recorded at 50, 100 and 125 MHz with the samespectrometers in deuteriochloroform. Chemical shifts (δ) were reporteddownfield from internal Me₄Si (δ 0.00). Electron impact (EI) massspectra were obtained with a Micromass AutoSpec (Beverly, Mass.)instrument. High-performance liquid chromatography (HPLC) was performedon a Waters Associates liquid chromatograph equipped with a Model 6000Asolvent delivery system, a Model U6K Universal injector, and a Model 486tunable absorbance detector. THF was freshly distilled before use fromsodium benzophenone ketyl under argon.

Example I Preparation of (20S)-2-methylene-25-hydroxy-19-nor-vitamin D₃(12, 1-desoxy-2MD) from the phosphine oxide 10

(a) α-Hydroxylation of a ketal-ketone 1 (SCHEME I).(R)-7-Hydroxy-1,4-dioxa-spiro[4.5]decan-8-one (2). To a stirred solutionof 1,4-cyclohexanedione monoethylene ketal (1; 3.00 g, 19.23 mmol) andL-proline (0.97 g, 8.42 mmol) in CHCl₃ (10 mL), a solution ofnitrosobenzene (3.60 g, 33.65 mmol) in CHCl₃ (16 mL) was slowly added at4° C. over 24 h by a syringe pump. Then the mixture was stirred at roomtemperature for additional 2 h. Reaction was quenched with brine, andthe organic materials were extracted with ethyl acetate, dried (MgSO₄)and concentrated in vacuum. Purification by column chromatography onsilica (0.5→20% ethyl acetate/hexane gradient) gave an oily α-hydroxyketone 2 (1.45 g, 44%). Purity of the product was checked by HPLC (4.6mm×25 cm Chiralcell OD-H column, 1.5 mL/min) using hexane/2-propanol(99:1) solvent system: it was found to have enantiomeric excess (ee)higher than 94% (R_(V)=5.7 mL; for the S-enantiomer R_(V)=4.7 mL).

2: [α]_(D)+27° (c 0.65, CHCl₃); ¹H NMR (200 MHz, CDCl₃) δ 1.85 (1H, t,J=12.4 Hz, 6β-H), 2.05 (2H, m, 10-H₂), 2.50 (br m, 6α- and 9β-H), 2.70(1H, dt, J=6.8, 13.2 Hz, 9α-H), 3.46 (1H, s, OH), 4.03 (4H, m,O—CH₂CH₂—O), 4.38 (1H, dd, J=12.4, 6.8 Hz, 7α-H); HRMS (ESI) exact masscalculated for C₈H₁₂O₄Na (M⁺+Na) 195.0633, found 195.0628.

(b) Protection of α-hydroxy ketone 2.(R)-7-[(tert-Butyldiphenylsilyl)oxy]-1,4-dioxa-spiro[4.5]decan-8-one(3). tert-Butyldiphenylsilyl chloride (3.55 mL, 3.75 g, 13.65 mmol) wasadded to a solution of α-hydroxy ketone 2 (1.60 g, 13.65 mmol) andimidazole (2.32 g, 33.9 mmol) in anhydrous DMF (9 mL). The mixture wasstirred at room temperature for 18 h. The reaction was quenched withbrine and extracted with hexane. The combined organic phases were dried(MgSO₄) and concentrated under reduced pressure. Column chromatographyon silica (1→4% hexane/ethyl acetate gradient) provided the protectedcompound 3 (3.35 g, 88%) as a colorless oil.

3: ¹H NMR (200 MHz, CDCl₃) δ 1.10 (9H, s, Si-t-Bu), 1.8-2.1 (4H, br m,6- and 10-H₂), 2.35 (2H, m, 9-H₂), 3.62 (1H, m, one of —O—CH₂CH₂—O—),3.82 (3H, m, three of —O—CH₂CH₂—O—), 4.40 (1H, dd, J=11.8, 7.6 Hz,7α-H), 7.38 (6H, m, Ar—H), 7.67 (4H, m, Ar—H); ¹³C NMR (50 MHz, CDCl₃) δ19.4, 27.1, 34.7, 35.9, 43.8, 64.5, 64.7, 73.8, 107.6, 127.8, 129.9,133.3, 134.1, 136.0, 207.7; HRMS (ESI) exact mass calculated forC₂₄H₃₀O₄SiNa (M⁺+Na) 433.1811, found 433.1800.

(c) Wittig reaction of the ketone 3.(R)-7-[(tert-Butyldiphenylsilyl)oxy]-8-methylene-1,4-dioxa-spiro[4.5]decane(4). To methyltriphenylphosphonium bromide (2.5 g, 6.99 mmol) inanhydrous THF (20 mL) at 0° C. was added dropwise n-BuLi (1.6 M inhexanes; 8.8 mL, 14.08 mmol). After 15 min another portion ofphosphonium salt (2.5 g, 6.99 mmol) was added, and the solution wasstirred at 0° C. for 10 min, and at room temperature for 20 min. Theorange-red mixture was then cooled to −78° C. and siphoned to theprecooled (−78° C.) solution of the ketone 3 (2.85 g, 6.93 mmol) inanhydrous THF (7 mL). The reaction mixture was stirred at −78° C. for 4h and then at room temperature for 1 h. The mixture was poured intobrine and extracted with hexane. Organic extracts were dried (MgSO₄),and evaporated to give an orange oily residue which was purified byflash chromatography on silica. Elution with hexane/ethyl acetate (97:3)gave pure 4-methylene compound 4 (2.62 g, 93%) as a colorless oil.

4: ¹H NMR (200 MHz, CDCl₃) δ 1.01 (9H, s, Si-t-Bu), 1.43 (2H, m, 10-H₂),1.62 (2H, m, 6-H₂), 2.19 (2H, m, 9-H₂), 3.36 (1H, m, one of O—CH₂CH₂—O),3.73 (3H, m, three of O—CH₂CH₂—O), 4.30 (1H, dd, J=11.0, 5.0 Hz, 7α-H),4.88 and 5.31 (1H and 1H, each br s, ═CH₂), 7.35 (6H, m, Ar—H), 7.70(4H, m, Ar—H); ¹³C NMR (50 MHz, CDCl₃) δ 14.5, 19.5, 22.9, 30.0, 31.8,36.3, 44.5, 64.1, 64.3, 71.2, 106.6, 109.2, 127.7, 129.8, 134.1, 134.8,135.9, 136.2, 149.3; HRMS (ESI) exact mass calcd for C₂₅H₃₂O₃SiNa(M⁺+Na) 431.2019, measured 431.2028.

(d) Deprotection of a carbonyl group in the ketal 4.(R)-3-[(tert-Butyldiphenylsilyl)oxy]-4-methylene-cyclohexanone (5). To asolution of ketal 4 (160 mg, 0.392 mmol) in methylene chloride (11 mL)at room temperature FeCl₃×6H₂O (547 mg, 2.02 mmol) was added. Theresulting dark yellow suspension was stirred for 50 min and quenched bythe addition of water. The aqueous layer was extracted with methylenechloride, the combined organic layers were dried (MgSO₄) andconcentrated under reduced pressure. Column chromatography on silica ofthe resulting yellow residue using hexane/ethyl acetate (95:5) yieldedketone 5 (141 mg, 99%) as a colorless oil.

5: ¹H NMR (500 MHz, CDCl₃): δ 1.05 (9H, s, Si-t-Bu), 2.32-2.52 (5H, brm, 2β-H, 5-H₂ and 6-H₂), 2.83 (1H, m, 2α-H), 4.47 (1H, br t, J˜6 Hz,3α-H), 4.90 (2H, s, ═CH₂), 7.40 (6H, m, Ar—H), 7.65 (4H, m, Ar—H); ¹³CNMR (125 MHz, CDCl₃) δ 19.6, 27.1, 32.8, 36.9, 44.8, 72.8, 107.1, 108.2,128.0, 129.8, 133.0, 133.3, 135.1, 207.7; HRMS (ESI) exact masscalculated for C₂₃H₂₈O₂SiNa (M⁺+Na) 387.1757, found 387.1746.

(e) Peterson reaction of the ketone 5.[(R)-3′-[(tert-Butyldiphenylsilyl)oxy]-4′-methylene-cyclohexylidene]aceticacid methyl ester (mixture of 6 and 7). To a solution ofdiisopropylamine (48.5 μL, 376 μmol) in anhydrous THF (260 μL) was addedn-BuLi (2.5 M in hexanes; 148 μL, 367 μmol) under argon at −78° C. withstirring, and methyl(trimethylsilyl)acetate (60 μL, 367 μmol) was thenadded. After 15 minutes keto compound 5 (63 mg, 172.8 μmol) in anhydrousTHF (300 μL+80 μL) was added dropwise. The solution was stirred at −78°C. for 2 hours, and the reaction was quenched with saturated NH₄Cl,poured into brine and extracted with ethyl acetate. The combined organicextracts were dried (MgSO₄) and evaporated. The residue was dissolved inhexane and applied on a silica Sep-Pak cartridge. Elution withhexane/ethyl acetate (98:2) gave unsaturated esters 6 and 7 (65 mg, 90%)as a colorless oil.

6 and 7 (mixture of isomers): ¹H NMR (200 MHz, CDCl₃; selected signals)δ 1.15 and 1.17 (5H and 4H, each s, 2×Si-t-Bu), 2.1-3.3 (6H, br m, 2′-,5′- and 6′-H₂), 3.69 and 3.73 (1.67H and 1.33H, each s, 2×COOCH₃), 4.29(1H, m, 3′α-H), 4.82, 4.90, 4.93, 5.12 (0.56H, 0.56H, 0.44H and 0.44H,each br s, ═CH₂), 5.48 and 5.83 (0.44H and 0.56H, each br s, CH—COOMe),7.45 (6H, m, Ar—H), 7.75 (4H, m, Ar—H); HRMS (ESI) exact mass calculatedfor C₂₆H₃₂O₃SiNa (M⁺+Na) 443.2019, found 443.2035.

(f) Reduction of the esters 6 and 7. (E)- and(Z)-2-[(R)-3′-[(tert-Butyldiphenylsilyl)oxy]-4′-methylene-cyclohexylidene]ethanols(8 and 9). Diisobutylaluminium hydride (1.5 M in toluene; 1.9 mL, 2.85mmol) was slowly added to a stirred solution of allylic esters 6 and 7(165 mg, 0.392 mmol) in toluene:methylene chloride (2:1; 8 mL) at −78°C. under argon. Stirring was continued at −78° C. for 1 h and at −40° C.for 30 min. The mixture was quenched by slow addition ofpotassium-sodium tartate (2N, 4 mL), aqueous HCl (2N, 4 mL) and H₂O (14mL), and extracted with ethyl acetate. Combined organic layers werewashed with brine, dried (MgSO₄) and evaporated. The residue was passedthrough a silica Sep-Pak cartridge with hexane/ethyl acetate (9:1). Theobtained mixture of allylic alcohols was separated by HPLC (9.4 mm×25 cmZorbax-Sil column, 4 mL/min) using hexane/ethyl acetate (8:2) solventsystem: the Z-isomer 9 (82 mg, 53%) was collected at R_(V)=35 mL and theE-isomer 8 (60 mg, 39%) at R_(V)=41 mL.

8 (minor E-isomer): ¹H NMR (500 MHz, CDCl₃) δ 1.08 (9H, s, Si-t-Bu),1.96 (1H, ˜dt, J˜5, 12.5 Hz, 6′β-H), 2.07 (1H, dd, J=12.5, 8.5 Hz,2′β-H), 2.08 (1H, m, 5′α-H), 2.13 (1H, dd, J=12.5, 4.5 Hz, 2′α-H), 2.31(1H, dt, J=12.5, 4.5 Hz, 6′α-H), 2.48 (1H, dt, J=12.5, 5.5 Hz, 5′β-H),4.09 (2H, d, J=7.0 Hz, —CH ₂OH), 4.14 (1H, dd, J=8.5, 4.5 Hz, 3′α-H),4.82 and 5.10 (1H and 1H, each br s, ═CH₂), 5.16 (1H, t, J=7.5 Hz, 2-H),7.39 (6H, m, Ar—H), 7.65 (4H, m, Ar—H); ¹³C NMR (125 MHz, CDCl₃) δ 19.3,27.0, 29.3, 32.7, 46.6, 58.7, 74.0, 107.2, 123.6, 127.5, 129.6, 133.8,134.5, 135.8, 139.7, 149.6; HRMS (ESI) exact mass calculated forC₂₅H₃₂O₂SiNa (M⁺+Na) 415.2070, found 415.2059.

9 (major Z-isomer): ¹H NMR (500 MHz, CDCl₃) δ 1.09 (9H, s, Si-t-Bu),1.99 (2H, m, 2′β- and 5′α-H), 2.11 (2H, m, 6′α- and 6′β-H), 2.25 (1H,dd, J=13.0, 4.5 Hz, 2′α-H), 2.48 (1H, dt, J=12.5, 5.5 Hz, 5′β-H), 3.62(1H, dd, J=10.0, 7.2 Hz, one of —CH ₂OH), 3.71 (1H, dd, J=10.0, 7.0 Hz,one of —CH ₂OH), 4.09 (1H, dd, J=9.0, 4.5 Hz, 3′α-H), 4.82 and 5.10 (1Hand 1H, each br s, ═CH₂), 5.37 (1H, t, J=7.0 Hz, 2-H), 7.39 (6H, m,Ar—H), 7.65 (4H, m, Ar—H); ¹³C NMR (125 MHz, CDCl₃) δ 19.3, 27.0, 33.4,37.3, 38.8, 58.3, 73.7, 107.1, 123.6, 127.6, 129.7, 133.7, 134.5, 135.8,139.4, 149.6; HRMS (ESI) exact mass calculated for C₂₅H₃₂O₂SiNa (M⁺+Na)415.2070, found 415.2067.

(g) Preparation of the phosphine oxide 10.[2-[(Z)—(R)-3′-[(tert-Butyldiphenylsilyl)oxy]-4′-methylene-cyclohexylidene]ethyl]diphenylphosphine oxide (10). To a solution of an allylic alcohol 8 (49 mg, 125μmol) in anhydrous THF (1.2 mL) was added n-BuLi (2.5 M in hexanes; 50μl, 125 μmol) under argon at 0° C. A solution of a freshlyrecrystallized tosyl chloride (24 mg, 125 μmol) in anhydrous THF (230μL) was then added to the allylic alcohol—n-BuLi solution. The mixturewas stirred at 0° C. for 5 min and set aside at 0° C. In another dryflask with air replaced by argon, n-BuLi (2.5 M in hexanes; 1 mL, 0.25mmol) was added to a solution of Ph₂PH (44.2 μl, 254 μmol) in anhydrousTHF (360 μL) at 0° C. with stirring. The red solution was siphoned underargon pressure to the solution of tosylate until the orange colorpersisted (ca. one-half of the solution was added). The resultingmixture was stirred for an additional 30 min at 0° C. and quenched byaddition of H₂O (14 μL). Solvents were evaporated under reducedpressure, the residue was redissolved in methylene chloride (1.2 mL),and stirred with 10% H₂O₂ (0.9 mL) at 0° C. for 1 h. The organic layerwas separated, washed with cold aqueous sodium sulfite and water, dried(MgSO₄), and evaporated. The residue was subjected to flashchromatography on silica. Elution with hexane/ethyl acetate (6:4) gavethe phosphine oxide 10 (64 mg, 79%).

10: ¹H NMR (200 MHz, CDCl₃) δ 1.08 (9H, s, Si-t-Bu), 1.35-2.45 (6H, brm, 2′-, 5′- and 6′-H₂), 2.52 (2H, br m, ═CH—CH ₂), 3.88 (1H, dd, J=10.0,5.0 Hz, 3′α-H), 4.80 and 5.17 (1H and 1H, each br s, ═CH₂), 5.14 (1H, m,2-H), 7.2-7.5 (16H, br m, Ar—H), 7.57 (2H, dd, J=8.0, 1.5 Hz, Ar—H),7.68 (2H, dd, J=8.0, 1.5 Hz, Ar—H); HRMS (ESI) exact mass calculated forC₃₇H₄₁O₂PSiNa (M⁺+Na) 599.2512, found 599.2534.

(h) Wittig-Horner reaction of the phosphine oxide 10 and the Grundmannketone 11. (20S)-25-hydroxy-2-methylene-19-norvitamin D₃ (12). To asolution of the phosphine oxide 10 (19 mg, 29.4 μmol) in anhydrous THF(230 μL) at 0° C. was slowly added n-BuLi (2.5 M in hexanes; 12 μL, 29.7μmol) under argon with stirring. The solution turned red. The mixturewas cooled to −78° C., and precooled (−78° C.) solution of protectedhydroxy ketone 11 (3 mg, 7.62 μmol) in anhydrous THF (60 μL+40 μL) wasslowly added. The mixture was stirred under argon at −78° C. for 1 h andat 0° C. for 19 h. Ethyl acetate was added, and the organic layer waswashed with brine, dried (MgSO₄) and evaporated. The residue wasdissolved in hexane, applied on a silica Sep-Pak cartridge and washedwith hexane/diethyl ether (98:2) to give the silylated 19-norvitaminderivative 12 (4 mg, 70%).

The product was dissolved in THF (350 μL) and tetrabutylammoniumfluoride (1.0 M in THF; 318 μL, 318 μmol) was added under argon at roomtemperature. The stirring was continued for 18 h, brine was added andthe mixture was extracted with ethyl acetate. The organic extracts weredried (MgSO₄) and evaporated. The residue was purified by HPLC (9.4mm×25 cm Zorbax-Sil column, 4 mL/min) using hexane/2-propanol acetate(95:5) solvent system; 19-norvitamin 12 (1.8 mg, 85%) was collected atR_(V)=19 mL. Analytical sample of the vitamin was obtained after HPLC(9.4 mm×25 cm Zorbax Eclipse XDB-C18 column, 4 mL/min) usingmethanol/water (85:15) solvent system (R_(V)=44 mL).

12: UV (EtOH) λ_(max) 244, 252, 261 nm; ¹H NMR (500 MHz, CDCl₃) δ 0.558(3H, s, 18-H₃), 0.857 (3H, d, J=6.5 Hz, 21-H₃), 1.217 (6H, s, 26- and27-H₃), 1.95-2.05 (2H, m), 2.14 (1H, m), 2.23-2.35 (2H, m), 2.37-2.47(2H, m), 2.59 (1H, dd, J=13.0, 4.1 Hz, 4α-H), 2.82 (1H, br dd, J˜13, 4.5Hz, 9β-H), 4.19 (1H, narr m, w/2=14 Hz, 3α-H), 4.83 and 4.96 (1H and 1H,each br s, ═CH₂), 5.84 and 6.21 (1H and 1H, each d, J=11.3 Hz, 7- and6-H); ¹³C NMR (100 MHz, CDCl₃) δ 12.3, 18.6, 20.9, 22.1, 23.5, 27.3,28.8, 29.1, 29.3, 32.2, 35.4, 36.0, 40.4, 44.3, 45.7, 46.7, 56.1, 56.3,71.1, 73.0, 107.2, 115.6, 121.4, 134.3, 142.3, 150.3; HRMS (ESI) exactmass calculated for C₂₇H₄₄O₂Na (M⁺+Na) 423.3239, found 423.3253.

Example II

Preparation of (20S)-2-methylene-25-hydroxy-19-nor-vitamin D₃ (12,1-desoxy-2MD) from the phosphine oxide 27.

(a) Lactonization of the quinic acid 13 (SCHEME II).(1S,3R,4R,5R)-1,3,4-Trihydroxy-6-oxa-bicyclo[3.2.1]octan-7-one (14). Asolution of D-(−)-quinic acid (20.0 g, 104 mmol) and p-toluenesulfonicacid (2.2 g, 11.6 mmol) in anhydrous toluene (200 mL) and anhydrous DMF(75 mL) was refluxed under Dean-Stark trap for 34 h. The reactionmixture was cooled to 23° C. and concentrated under reduced pressure toafford a thick yellow oil. It was diluted with methylene chloride (100mL), hexane (200 mL) was added and the resulting mixture was set asideat 23° C. for 12 h. The precipitated product was collected by vacuumfiltration, and it was further dried in vacuo to afford the lactone 14(13.0 g, 72%) as a white powder (mp 184-188° C., lit. mp 184-185° C.).

14: ¹H NMR (500 MHz, CD₃OD) δ 1.87 (1H, br t, J˜11 Hz, 2α-H), 2.02 (1H,ddd, J=11.7, 6.5, 2.9 Hz, 2β-H), 2.22 (1H, ddd, J=11.4, 6.0, 2.9 Hz,8β-H), 2.47 (1H, d, J=11.4 Hz, 8α-H), 3.70 (1H, ddd, J=11.4, 6.5, 4.4Hz, 3β-H), 3.98 (1H, br t, J˜5 Hz, 4β-H), 4.70 (1H, br t, J˜5 Hz, 5α-H);¹³C NMR (125 MHz, CD₃OD) δ 37.8, 40.0, 66.8, 67.3, 73.1, 77.8, 179.5;HRMS (ESI) exact mass calculated for C₇H₁₀O₅Na (M⁺+Na) 197.0426, found197.0420.

(b) Hydroxyl groups protection in the lactone 14.(1R,2S,6R,8R)-8-Hydroxy-4-thioxo-3,5,10-trioxa-tricyclo[6.2.1.0*2,6*]undecan-9-one(15). 1,1′-Thiocarbonyldiimidazole (1.3 g, 6.86 mmol) was added to asuspension of the lactone 14 (1.08 g, 6.24 mmol) in anhydrousacetonitrile (70 mL). The mixture was stirred vigorously at roomtemperature for 6 h and then solvent was evaporated under reducedpressure. The residue was purified by column chromatography on silica(3% methanol/methylene chloride) to give the tricyclic compound 15 (0.84g, 63%) as colorless crystals (mp 219-222° C.).

15: [α]_(D) −9.7° [c 1.06, (CH₃)₂CO]; ¹H NMR [400 MHz, (CD₃)₂CO] δ 2.24(2H, m), 2.62 (1H, m), 2.68 (1H, ddd, J=15.2, 8.0, 2.5 Hz, 7α-H), 5.03(1H, dd, J=5.9, 2.7 Hz, 1α-H), 5.26 (1H, m, 2β-H), 5.56 (1H, dt, J=3.1,8.0 Hz, 6β-H); ¹³C NMR [100 MHz, (CD₃)₂CO] δ 35.5, 37.8, 70.7, 73.3,77.7, 79.4, 176.8, 191.3; MS (EI) m/z (relative intensity) 216 (M⁺, 60),211 (15), 204 (12), 196 (42), 181 (100); exact mass calculated forC₈H₈O₅S 216.0092, found 216.0089.

(c) Reduction of compound 15 with tri-n-butyltin hydride.(1S,3S,5S)-1,3-Dihydroxy-6-oxa-bicyclo[3.2.1]octan-7-one (16) and(1S,4R,5R)-1,4-dihydroxy-6-oxa-bicyclo[3.2.1]octan-7-one (17).Tri-n-butyltin hydride (7.88 mL; 8.55 g, 29.40 mmol) was added bysyringe pump within 75 min to a refluxing solution of compound 15 (3.18g, 14.7 mmol) and 2,2′-azobisisobutyronitrile (0.36 g, 2.20 mmol) inanhydrous benzene/THF (3/1, 230 mL). The mixture was heated under refluxfor further 3 h and then set aside for 12 h. The solvents wereevaporated under reduced pressure and the residue was purified by columnchromatography on silica (15→40% acetone/methylene chloride gradient) togive the diol 17 (1.94 g, 83%) as colorless crystals (mp 212-214° C.)and the isomeric compound 16 (0.11 g, 5%) as colorless crystals (mp148-152° C.).

16: [α]_(D) −60.3° (c 0.84, CH₃OH), lit [α]_(D) −59.0° (c 0.80, CH₃OH);¹H NMR [400 MHz, (CD₃)2CO] δ1.48 (1H, dd, J=13.4, 10.0 Hz, 4α-H), 1.74(1H, t, J˜11 Hz, 2α-H), 2.02 (1H, d, J=11.1 Hz, 8α-H), 2.23 (1H, m),2.34 (1H, m), 2.42 (1H, m), 3.88 (1H, m, 3β-H), 4.78 (1H, t, J˜5 Hz,5α-H); ¹H NMR (400 MHz, CD₃OD) δ 1.44 (1H, dd, J=13.4, 10.1 Hz, 4α-H),1.75 (1H, t, J˜11 Hz, 2α-H), 1.97 (1H, d, J=11.2 Hz, 8α-H), 2.23 (1H,m), 2.36-2.43 (2H, m), 3.87 (1H, m, 3β-H), 4.81 (1H, t, J˜5 Hz, 5α-H);¹³C NMR (100 MHz, CD₃OD) δ 37.3, 44.3, 44.4, 65.3, 74.1, 75.9. 179.7;HRMS (ESI) exact mass calculated for C₇H₁₀O₄Na (M⁺+Na) 181.0477, found181.0482.

17: [α]_(D) −59.7° [c 1.15, (CH₃)₂ CO]; ¹H NMR (400 MHz, CD₃OD) δ 1.70(1H, m), 1.78 (1H, m), 1.86 (1H, m), 1.97 (1H, m), 2.26 (1H, ddd,J=11.3, 6.1, 2.7 Hz, 8β-H), 2.45 (1H, d, J=11.3 Hz, 8α-H), 3.97 (1H, brt, J˜4 Hz, 4β-H), 4.60 (1H, br t, J˜5 Hz, 5α-H); ¹H NMR [400 MHz,(CD₃)₂CO] δ 1.66-1.77 (2H, m), 1.84 (1H, m), 1.96 (1H, m), 2.24 (1H,ddd, J=11.2, 6.2, 2.8 Hz, 8β-H), 2.46 (1H, d, J=11.2 Hz, 8α-H), 4.00(1H, m, 4β-H), 4.58 (1H, br t, J˜5 Hz, 5α-H); ¹³C NMR [100 MHz,(CD₃)₂CO] δ 28.1, 30.2, 38.2, 64.4, 74.7, 77.6, 178.3; HRMS (ESI) exactmass calculated for C₇H₁₀O₄Na (M⁺+Na) 181.0477, found 181.0471.

(d) Oxidation of the diol 17.(1S,5R)-1-Hydroxy-6-oxa-bicyclo[3.2.1]octane-4,7-dione (18). The mixtureof alcohol 17 (2.67 g, 16.88 mmol), oven-dried activated 4 Å molecularsieves (2.7 g), pyridinium dichromate (12.90 g, 34.28 mmol) andanhydrous acetonitrile (250 mL) was stirred vigorously at roomtemperature for 5 h. The reaction mixture was then filtered through apad of Celite (washed with 300 mL of ethyl acetate) and the solventswere removed under reduced pressure. Column chromatography of theresidue on silica (15→40% acetone/methylene chloride gradient) affordedthe ketone 18 (2.17 g, 82%) as colorless crystals (mp 144-145° C.).

18: [α]_(D) −225° [c 1.16, (CH₃)₂CO]; ¹H NMR (500 MHz, CD₃CN) δ 2.11(2H, m, 2-H₂), 2.35 (1H, d, J=12.5 Hz, 8α-H), 2.46 (1H, br dd, J=16.7,˜6 Hz, 3α-H), 2.70 (1H, dt, J=16.7, ˜10 Hz, 3β-H), 2.78 (1H, ddd,J=12.5, 6.8, 2.8 Hz, 8β-H), 4.51 (1H, d, J=6.8 Hz, 5α-H); ¹³C NMR (125MHz, CD₃CN) δ 32.8, 34.0, 42.6, 74.4, 80.5, 178.0, 204.7; MS (EI) m/z156 (M⁺, 55), 138 (100), 100 (42), 70 (43); HRMS (EI) exact masscalculated for C₇H₈O₄ 156.0423, found 156.0417.

(e) Wittig reaction of the ketone 18.(1S,5R)-1-Hydroxy-4-methylene-6-oxa-bicyclo[3.2.1]octan-7-one (19). Tomethyl triphenylphosphonium bromide (1.78 g, 4.99 mmol) in anhydrous THF(35 mL) at 0° C. a solution of potassium tert-butoxide in THF (1.0 M;4.74 mL, 4.74 mmol) was added dropwise. The mixture was warmed up toroom temperature and stirred for additional 10 min. Ketolactone 18 (0.38g, 2.43 mmol) in THF (10 mL) was added via cannula and stirring wascontinued at room temperature for next 2 h. The solvent was then removedunder reduced pressure and the residue was partitioned between ethylacetate (20 mL) and brine (40 mL). The layers were separated and theaqueous layer was extracted with ethyl acetate (5×20 mL). The organicextracts were combined, dried (MgSO₄), filtered and concentrated underreduced pressure. The residue was purified by column chromatography onsilica (30% ethyl acetate/hexane) to give the semisolid compound 19(0.30 g, 80%).

19: [α]_(D) −129° (c 1.15, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 1.96 (2H,m), 2.05 (1H, d, J=11.3 Hz, 8α-H), 2.48 (2H, m), 2.70 (1H, ddd, J=11.3,6.3, 2.7 Hz, 8β-H), 4.93 (1H, br s, one of ═CH₂), 4.98 (1H, d, J=6.3 Hz,5α-H), 5.01 (1H, d, J=1.4 Hz, one of ═CH₂); ¹³C NMR (100 MHz, CDCl₃) δ26.3, 33.8, 44.1, 74.4, 80.2, 112.1, 141.4, 178.7; HRMS (ESI) exact masscalculated for C₈H₁₀O₃Na (M⁺+Na) 177.0528, found 177.0525.

(f) Methanolysis of the lactone 19 and hydroxyl protection.[(1S,3R)-3-[(tert-Butyldimethylsilyl)oxy]-1-hydroxy-4-methylene]cyclohexanecarboxylicacid methyl ester (20). The lactone 19 (0.88 g, 5.71 mmol) was treatedwith anhydrous methanol (30 mL) in a presence of activated, oven-dried 4Å molecular sieves (0.22 g). The reaction mixture was stirred at roomtemperature for 48 h. Then molecular sieves were filtered out and thesolvent was evaporated under reduced pressure. The crude methyl esterwas dissolved in anhydrous methylene chloride (30 mL) and 2,6-lutidine(1.26 mL; 1.16 g, 10.84 mmol), cooled to −40° C. andtert-butyldimethylsilyl trifluoromethanesulfonate (1.97 mL; 2.26 g, 8.56mmol) was added dropwise. The reaction mixture was stirred at −40° C.for 50 min. Wet methylene chloride (10 mL) was added slowly, coolingbath was removed and the reaction mixture was allowed to warm up slowlyto room temperature. Then it was filtered through a pad of Celite(washed with 30 mL of methylene chloride), washed with saturated aqueousCuSO₄ (2×15 mL) and brine (15 mL). The organic layers were combined,dried (MgSO₄), filtered and concentrated under reduced pressure. Thecrude product was purified by column chromatography on silica (10% ethylacetate/hexane) to afford ester 20 (1.37 g, 80%) of as a colorless oil.

20: [α]_(D) −0.2° (c 1.05, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 0.07 and0.08 (3H and 3H, each s, 2×SiCH₃), 0.90 (9H, s, Si-t-Bu), 1.70 (1H, m),1.81 (2H, m), 1.97 (1H, m), 2.33 (1H, ddd, J=13.5, 4.3, 2.4 Hz, 5β-H),2.46 (1H, dt, J=13.5, 4.3 Hz, 5α-H), 3.77 (3H, s, COOCH₃), 4.42 (1H, dd,J=11.1, 5.0 Hz, 3α-H), 4.80 and 5.03 (1H and 1H, each s, ═CH₂); ¹³C NMR(125 MHz, CDCl₃) δ −5.05, −5.01, 18.3, 25.8, 29.3, 36.0, 45.3, 53.0,69.0, 75.1, 105.7, 149.6, 176.6; HRMS (ESI) exact mass calculated forC₁₅H₂₈O₄SiNa (M⁺+Na) 323.1655, found 323.1643.

(g) Reduction of the ester 20.[(1S′,3R′)-3′-[(tert-Butyldimethylsilyl)oxy]-1′-hydroxy-4′-methylene-cyclohexyl]methanol(21). Diisobutylaluminium hydride (1.0 M in methylene chloride; 3.83 mL,3.83 mmol) was slowly added to a stirred solution of methyl ester 20(0.23 g, 0.76 mmol) in methylene chloride (18 mL) at −70° C. Stirringwas continued at −70° C. for 1 h and at −30° C. for 2 h. The mixture wasquenched by slow addition of potassium-sodium tartrate (2 N, 4 mL),diluted with methylene chloride (200 mL), washed with brine (3×30 mL)and water (2×30 mL). Organic layer was dried (MgSO₄), filtered andconcentrated under reduced pressure. The residue was passed through asilica Sep-Pak cartridge (10→30% ethyl acetate/hexane gradient) to givethe diol 21 (0.20 g, 94%) as colorless crystals (mp 89-90° C.).

21: [α]_(D) +1.3° (c 1.04, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 0.07 and0.08 (3H and 3H, each s, 2×SiCH₃), 0.91 (9H, s, Si-t-Bu), 1.40 (2H, m),1.70 (1H, m), 1.96 (1H, m), 2.33 (2H, m), 3.48 (2H, s, —CH ₂OH), 4.40(1H, dd, J=10.2, 5.0 Hz, 3′α-H), 4.78 and 4.97 (1H and 1H, each s,═CH₂); ¹³C NMR (125 MHz, CDCl₃) δ −5.0, −4.9, 18.3, 25.8, 28.9, 35.1,44.7, 69.7, 70.7, 73.4, 105.9, 150.4; HRMS (ESI) exact mass calculatedfor C₁₅H₂₈O₃Si (M⁺+H) 273.1886, found 273.1885.

(h) Periodate oxidation of the diol 21.(R)-3-[(tert-Butyldimethylsilyl)oxy]-4-methylene-cyclohexanone (22). Toa stirred solution of diol 21 (0.76 g, 2.81 mmol) in methanol/water(5/1, 53 mL) was added sodium periodate (1.80 g, 8.43 mmol) at 0° C.Stirring was continued at 0° C. for 1 h, the reaction mixture wasdiluted with ethyl acetate (140 mL) and extracted with brine (3×20 mL)and water (20 mL). The combined aqueous layers were extracted with ethylacetate (3×20 mL). The organic layers were combined and dried (MgSO₄),then filtered and concentrated under reduced pressure. The residue waspurified on a silica Sep-Pak cartridge (5% ethyl acetate/hexane) to givethe ketone 22 (0.64 g, 95%) as a colorless oil.

22: [α]_(D) −43° (c 1.25, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 0.03 and0.05 (3H and 3H, each s, 2×SiCH₃), 0.86 (9H, s, Si-t-Bu), 2.28-2.45 (3H,m), 2.48 (1H, dd, J=14.1, 6.7 Hz, 2β-H), 2.60 (1H, dd, J=14.1, 4.2 Hz,2α-H), 2.71 (1H, m), 4.42 (1H, m, 3β-H), 4.94 and 5.08 (1H and 1H, eachs, ═CH₂); ¹³C NMR (100 MHz, CDCl₃) δ −5.1, −5.0, 18.1, 25.6, 28.7, 41.5,51.5, 72.7, 109.8, 147.0, 208.9; HRMS (ESI) exact mass calculated forC₁₃H₂₄O₂SiNa (M⁺+Na) 263.1443, found 263.1438.

(i) Wittig reaction of the ketone 22.[(R)-3′-[(tert-Butyldimethylsilyl)oxy]-4′-methylene-cyclohexylidene]aceticacid methyl ester (mixture of 23 and 24). To a solution of ketone 22(0.65 g, 2.38 mmol) in anhydrous benzene (20 mL)methyl(triphenylphosphoranylidene) acetate (1.59 g, 4.77 mmol) wasadded. The reaction mixture was heated under reflux for 15 h, thenconcentrated under reduced pressure and the residue was purified on asilica Sep-Pak cartridge (3→5% ethyl acetate/hexane) to afford a mixtureof unsaturated esters 23 and 24 (ratio ca. 2:3; 0.63 g, 89%).

23 and 24 (mixture of isomers): ¹H NMR (400 MHz, CDCl₃; selectedsignals) δ 0.05 and 0.07 and 0.11 (1.2H, 3H and 1.8H, each s, 2×SiCH₃),0.90 (9H, s, Si-t-Bu), 3.29 (0.4H, m, 6′α-H), 3.51 (0.6H, dd, J=13.0,4.4 Hz, 2′α-H), 4.12 (1H, m, 3′α-H), 4.80, 4.82, 5.01 and 5.03 (0.6H,0.4H, 0.4H and 0.6H, each s, ═CH₂), 5.68 and 5.73 (0.4H and 0.6H, s,2-H); HRMS (ESI) exact mass calculated for C₁₆H₂₈O₃SiNa (M⁺+Na)319.1705, found 319.1710.

(j) Reduction of the esters 23 and 24. (E)- and(Z)-2-[(R)-3′-[(tert-Butyldimethylsilyl)oxy]-4′-methylene-cyclohexylidene]ethanols(25 and 26). Diisobutylaluminium hydride (1.0 M in methylene chloride;9.89 mL, 9.89 mmol) was slowly added to a stirred solution of themixture of the esters 23 and 24 (0.73 g, 2.47 mmol) in methylenechloride (40 mL) at −70° C. Stirring was continued at −70° C. for 2 hand −40° C. for 1 h. The mixture was quenched by slow addition ofpotassium-sodium tartrate (2 N, 6 mL), diluted with methylene chloride(250 mL), washed with brine (3×30 mL) and water (2×30 mL). Organic layerwas dried (MgSO₄), filtered and concentrated under reduced pressure. Theresidue was purified by column chromatography on silica (10→20% ethylacetate/hexane gradient) to give an allylic alcohol 25 (0.25 g, 39%) ofas a colorless oil and the oily isomeric compound 26 (0.37 g, 58%).

25 (minor E-isomer): [α]_(D) +5.8° (c 0.98, CHCl₃); ¹H NMR (400 MHz,CDCl₃) δ 0.06 and 0.07 (3H and 3H, each s, 2×SiCH₃), 0.91 (9H, s,Si-t-Bu), 1.97 (2H, m), 2.14 (1H, br t, J˜11 Hz, 2′β-H), 2.47 (3H, m),4.05 (1H, dd, J=9.8, 4.9 Hz, 3′α-H), 4.17 (2H, m, —CH ₂OH), 4.78 and5.01 (1H and 1H, each s, ═CH₂), 5.46 (1H, t, J=7.0 Hz, 2-H); ¹³C NMR(100 MHz, CDCl₃) δ −5.0, −4.9, 18.3, 25.8, 29.2, 32.9, 47.3, 58.7, 73.2, 106.5, 123.4, 140.2, 150.0; HRMS (ESI) exact mass calculated forC₁₅H₂₈O₂SiNa (M⁺+Na) 291.1756, found 291.1756.

26 (major Z-isomer): [α]_(D) −26° (c 1.09, CHCl₃); ¹H NMR (500 MHz,CDCl₃) δ 0.06 and 0.08 (3H and 3H, each s, 2×SiCH₃), 0.90 (9H, s,Si-t-Bu), 2.09 (1H, dt, J=12.7, 6.6 Hz, 5′β-H), 2.17 (2H, m, 5′α- and6′β-H), 2.27 (1H, dd, J=12.9, 7.9 Hz, 2′β-H), 2.43 (1H, dt, J=12.7, 5.8Hz, 6′α-H), 2.50 (1H, dd, J=12.9, 4.2 Hz, 2′α-H), 4.09 (1H, m, one of—CH ₂OH), 4.13 (1H, dd, J=7.9, 4.2 Hz, 3′α-H), 4.18 (1H, dd, J=11.9, 7.2Hz, one of —CH ₂OH), 4.78 and 4.96 (1H and 1H, each s, ═CH₂), 5.60 (1H,t, J=7.2 Hz, 2-H); ¹³C NMR (125 MHz, CDCl₃) δ −4.94, −4.9, 18.3, 25.8,33.1, 37.4, 39.0, 58.3, 73.3, 107.6, 123.7, 140.4, 149.9; HRMS (ESI)exact mass calculated for C₁₅H₂₈O₂SiNa (M⁺+Na) 291.1756, found 291.1769.

(k) Preparation of the phosphine oxide 27.[(E)-[(3′R)-[(tert-Butyldimethylsilyl)oxy]-4′-methylenecyclohexylidene]ethyl]diphenylphosphineOxide (27). To a allylic alcohol 25 (190.0 mg, 708 μmol) in anhydrousTHF (5 mL) was added n-BuLi (1.6 M in hexanes; 450 μL, 125 μmol) underargon at 0° C. A solution of a freshly recrystallized tosyl chloride(141.5 mg, 743 μmol) in anhydrous THF (1.5 mL) was then added to theallylic alcohol—n-BuLi solution. The mixture was stirred at 0° C. for 5min and set aside at 0° C. In another dry flask with air replaced byargon, n-BuLi (1.6 M in hexanes; 942 μL, 1.51 mmol) was added todiphenylphosphine (250 μL, 1.44 mmol) in anhydrous THF (2 mL) at 0° C.with stirring. The red solution was siphoned under argon pressure to thesolution of tosylate until the orange color persisted. The resultingmixture was stirred for an additional 30 min at 0° C. and quenched byaddition of H₂O (0.8 mL). Solvents were evaporated under reducedpressure, the residue was redissolved in methylene chloride (6.5 mL),and stirred with 10% H₂O₂ (3.8 mL) at 0° C. for 1 h. The organic layerwas separated, washed with cold aqueous sodium sulfite (1.5 mL) andwater (1.5 mL), dried (MgSO₄), filtered and evaporated under reducedpressure. The residue was purified on a silica Sep-Pak cartridge (5%ethyl acetate/hexane) to afford semicrystalline phosphine oxide 27 (220mg, 69%).

27: [α]_(D) +7.9° (c 1.07, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 0.03 (6H,s, 2×SiCH₃), 0.88 (9H, s, Si-t-Bu), 1.60 (2H, m), 2.04 (1H, m, 2′β-H),2.25 (2H, m), 2.39 (1H, dd, J=12.6, 4.8 Hz, 2′α-H), 3.12 (2H, m, ═CH—CH₂), 3.85 (1H, dd, J=9.8, 4.8 Hz, 3′α-H), 4.70 and 4.95 (1H and 1H, eachs, ═CH₂), 5.30 (1H, ˜t, J=7 Hz, 2-H), 7.25-7.5 (6H, m, Ar—H), 7.74 (4H,m, Ar—H); ¹³C NMR (125 MHz, CDCl₃) δ −5.0, 18.3, 25.8, 29.2, 30.1, 30.7,32.4, 47.4, 73.1, 106.2, 112.2, 112.3, 128.4, 128.5, 128.51, 128.55,131.0, 131.1, 131.2, 131.8, 141.2, 141.3, 149.9; HRMS (ESI) exact masscalculated for C₂₇H₃₇O₂PSiNa (M⁺+Na) 475.2198, found 475.2208.

(l) Wittig-Horner reaction of the phosphine oxide 27 and the Grundmannketone 11. (20S)-25-hydroxy-2-methylene-19-norvitamin D₃ (12). To astirred solution of 27 (49 mg, 108 μmol) in anhydrous THF (800 μL) 2drops of phenyl lithium solution (1.8 M in di-n-butyl ether) were addedat −20° C. until the solution became deep orange. Then 54 μL (97 μmol)of the of phenyl lithium solution was added dropwise. After 20 min thereaction mixture was cooled to −78° C. and the precooled (−78° C.)solution of ketone 11 (28 mg, 71 μmol) in anhydrous THF (400 μL+100 μL)was added via cannula. The mixture was stirred for 2 h at −78° C. and at0° C. for 4 h. Ethyl acetate (30 mL) was added and the organic phase waswashed with brine (7 mL), dried (MgSO₄), filtered and concentrated underreduced pressure. The residue was purified on a silica Sep-Pak cartridge(0→2% ethyl acetate/hexane) to give protected 19-norvitamin Dderivative.

To a stirred solution of the obtained oily compound (11 mg, 17 μmol) inmethanol (2 mL) (15)-(+)-10-camphorsulfonic acid (7 mg, 30 μmol) wasadded at 0° C. Then cooling bath was removed and the reaction mixturewas stirred overnight at room temperature. A few drops of saturatedaqueous solution of sodium bicarbonate and water (3 mL) was added andthe mixture was extracted with ethyl acetate (5×7 mL). The combinedorganic phases were dried (MgSO₄), concentrated under reduced pressureand the residue was purified on a silica Sep-Pak cartridge (10% ethylacetate/hexane) as well as by HPLC (9.4 mm×25 cm Zorbax Rx-Sil column, 4mL/min) using 7% hexane/2-propanol solvent system (R_(V)=21 mL). Furtherpurification was achieved by HPLC (9.4 mm×25 cm Zorbax Eclipse XDB-C18column, 4 mL/min) using methanol/water (85:15) solvent system (R_(V)=44mL). Analytically pure 19-norvitamin D₃ analog 12 (6.9 mg, 14% yieldfrom 11) was obtained, identical in all respects with the compounddescribed in the EXAMPLE I.

SCHEME I and SCHEME II are set forth below.

Biological Activity of(20S)-25-Hydroxy-1-Desoxy-2-Methylene-19-Norvitamin D₃

The introduction of a methylene group to the 2-position, the removal ofthe methylene substituent at carbon 10, and orienting the methyl groupat carbon 20 in its epi or S configuration had little or no effect onbinding to the full length recombinant rat vitamin D receptor, ascompared to 1α,25-dihydroxyvitamin D₃. The compound 1-desoxy-2MD boundwith only slightly less affinity to the receptor as compared to thestandard 1,25-(OH)₂D₃ (FIG. 1). It might be expected from these resultsthat compound 1-desoxy-2MD would have equivalent biological activity.Surprisingly, however, compound 1-desoxy-2MD is a highly selectiveanalog with unique biological activity.

FIGS. 5A and 5B show that 1-desoxy-2MD has relatively low activity ascompared to that of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), the naturalhormone, in stimulating intestinal calcium transport. 1-desoxy-2MD isabout 20 times less potent than 1,25(OH)₂D₃ in promoting active calciumtransport across the gut.

FIGS. 4A and 4B demonstrate that 1-desoxy-2MD has relatively low bonecalcium mobilization activity, as compared to 1,25(OH)₂D₃. 1-desoxy-2MDis less potent than the native hormone in releasing bone calcium storesas little to no activity is observed until 7020 pmol/rat isadministered; whereas, significant increases in serum calcium areobserved at 780 pmol when the native hormone is given.

FIGS. 4-5 thus illustrate that 1-desoxy-2MD may be characterized ashaving relatively low calcemic activity.

FIG. 2 illustrates that 1-desoxy-2MD is almost as potent as 1,25(OH)₂D₃on HL-60 cell differentiation, making it an excellent candidate for thetreatment of a cancer, especially for the prevention or treatment ofleukemia, colon cancer, breast cancer, skin cancer and prostate cancer.

FIG. 3 illustrates that the compound 1-desoxy-2MD has lesstranscriptional activity than 1α,25-dihydroxyvitamin D₃ in bone cells.In bone cells, 1-desoxy-2MD is about 20 times less potent than1,25(OH)₂D₃ in increasing transcription of the 24-hydroxylase gene. Thisresult, together with the cell differentiation activity of FIG. 2,suggests that 1-desoxy-2MD will be very effective in treating the abovereferred to cancers because it has direct cellular activity in causingcell differentiation, gene transcription, and in suppressing cellgrowth.

Experimental Methods

The compounds of the invention were prepared and studied using thefollowing methods.

Vitamin D Receptor Binding

Test Material

Protein Source

Full-length recombinant rat receptor was expressed in E. coli BL21 (DE3)Codon Plus RIL cells and purified to homogeneity using two differentcolumn chromatography systems. The first system was a nickel affinityresin that utilizes the C-terminal histidine tag on this protein. Theprotein that was eluted from this resin was further purified using ionexchange chromatography (S-Sepharose Fast Flow). Aliquots of thepurified protein were quick frozen in liquid nitrogen and stored at −80°C. until use. For use in binding assays, the protein was diluted inTEDK₅₀ (50 mM Tris, 1.5 mM EDTA, pH7.4, 5 mM DTT, 150 mM KCI) with 0.1%Chaps detergent. The receptor protein and ligand concentration wasoptimized such that no more than 20% of the added radiolabeled ligandwas bound to the receptor.

Study Drugs

Unlabeled ligands were dissolved in ethanol and the concentrationsdetermined using UV spectrophotometry (1,25(OH)₂D₃: molar extinctioncoefficient=18,200 and λ_(max)=265 nm). Radiolabeled ligand(³H-1,25(OH)₂D₃, ˜159 Ci/mmole) was added in ethanol at a finalconcentration of 1 nM.

Assay Conditions

Radiolabeled and unlabeled ligands were added to 100 mcl of the dilutedprotein at a final ethanol concentration of ≦10%, mixed and incubatedovernight on ice to reach binding equilibrium. The following day, 100mcl of hydroxylapatite slurry (50%) was added to each tube and mixed at10-minute intervals for 30 minutes. The hydroxylapaptite was collectedby centrifugation and then washed three times with Tris-EDTA buffer (50mM Tris, 1.5 mM EDTA, pH 7.4) containing 0.5% Titron X-100. After thefinal wash, the pellets were transferred to scintillation vialscontaining 4 ml of Biosafe II scintillation cocktail, mixed and placedin a scintillation counter. Total binding was determined from the tubescontaining only radiolabeled ligand.

HL-60 Differentiation

Test Material

Study Drugs

The study drugs were dissolved in ethanol and the concentrationsdetermined using UV spectrophotometry. Serial dilutions were prepared sothat a range of drug concentrations could be tested without changing thefinal concentration of ethanol (≦0.2%) present in the cell cultures.

Cells

Human promyelocytic leukemia (HL60) cells were grown in RPMI-1640 mediumcontaining 10% fetal bovine serum. The cells were incubated at 37° C. inthe presence of 5% CO₂.

Assay Conditions

HL60 cells were plated at 1.2×10⁵ cells/ml. Eighteen hours afterplating, cells in duplicate were treated with drug. Four days later, thecells were harvested and a nitro blue tetrazolium reduction assay wasperformed (Collins et al., 1979; J. Exp. Med. 149:969-974). Thepercentage of differentiated cells was determined by counting a total of200 cells and recording the number that contained intracellularblack-blue formazan deposits. Verification of differentiation tomonocytic cells was determined by measuring phagocytic activity (datanot shown).

In vitro Transcription Assay

Transcription activity was measured in ROS 17/2.8 (bone) cells that werestably transfected with a 24-hydroxylase (24Ohase) gene promoterupstream of a luciferase reporter gene (Arbour et al., 1998). Cells weregiven a range of doses. Sixteen hours after dosing the cells wereharvested and luciferase activities were measured using a luminometer.RLU=relative luciferase units.

Intestinal Calcium Transport and Bone Calcium Mobilization

Male, weanling Sprague-Dawley rats were placed on Diet 11 (Suda et al,J. Nutr. 100:1049, 1970) (0.47% Ca)+vitamins AEK for one week followedby Diet 11 (0.02% Ca)+vitamins AEK for 3 weeks. The rats were thenswitched to the same diet containing 0.47% Ca for one week followed bytwo weeks on the same diet containing 0.02% Ca. Dose administrationbegan during the last week on 0.02% calcium diet. Four consecutive ipdoses were given approximately 24 hours apart. Twenty-four hours afterthe last dose, blood was collected from the severed neck and theconcentration of serum calcium determined by atomic absorptionspectrometry as a measure of bone calcium mobilization. The first 10 cmof the intestine was also collected for intestinal calcium transportanalysis using the everted gut sac method.

Interpreation of Data

VDR binding, HL60 cell differentiation, and transcription activity.1-desoxy-2MD (K_(i)=1×10⁻¹⁰M) has slightly less activity than thenatural hormone 1α,25-dihydroxyvitamin D₃ (K_(i)=5×10⁻¹¹M) in itsability to compete with [³H]-1,25(OH)₂D₃ for binding to the full-lengthrecombinant rat vitamin D receptor (FIG. 1). 1-desoxy-2MD is also alittle less potent (EC₅₀=1×10⁻⁸M) in its ability (efficacy or potency)to promote HL60 differentiation as compared to 1α,25-dihydroxyvitamin D₃(EC₅₀=4×10⁻⁹M) (See FIG. 2). Also, compound 1-desoxy-2MD (EC₅₀=4×10⁻⁹M)has about 20 times less transcriptional activity in bone cells than1α,25-dihydroxyvitamin D₃ (EC₅₀=2×10⁻¹⁰M) (See FIG. 3). These data alsoindicate that 1-desoxy-2MD will have significant activity as ananti-cancer agent, especially for preventing or treating leukemia, coloncancer, breast cancer, skin cancer and prostate cancer because it hasdirect cellular activity in causing cell differentiation and insuppressing cell growth.

Calcium mobilization from bone and intestinal calcium absorption invitamin D-deficient animals. Using vitamin D-deficient rats on a lowcalcium diet (0.02%), the activities of 1-desoxy-2MD and 1,25(OH)₂D₃ inintestine and bone were tested. As expected, the native hormone(1,25(OH)₂D₃) increased serum calcium levels at the dosages tested(FIGS. 4A and 4B). FIGS. 4A and 4B also show that 1-desoxy-2MD hassignificantly less activity in mobilizing calcium from bone than1,25(OH)₂D₃. Administration of 1-desoxy-2MD at 780 pmol/day for 4consecutive days resulted in little or no mobilization of bone calcium.1-desoxy-2MD is less potent than the native hormone in releasing bonecalcium stores as little to no activity is observed until 7020 pmol/ratis administered; whereas, significant increases in serum calcium areobserved at 780 pmol when the native hormone is given.

Intestinal calcium transport was evaluated in the same groups of animalsusing the everted gut sac method (FIGS. 5A and 5B). These results showthat the compound 1-desoxy-2MD is about 20 times less potent inpromoting intestinal calcium transport activity when administered at therecommended lower dosages, as compared to 1,25(OH)₂D₃. Thus, it may beconcluded that 1-desoxy-2MD has low intestinal calcium transportactivity at the tested doses.

These results further illustrate that 1-desoxy-2MD is an excellentcandidate for numerous human therapies as described herein. 1-desoxy-2MDis an excellent candidate for treating a cancer because: (1) it hassignificant VDR binding, transcription activity and cellulardifferentiation activity; (2) it has low risk of hypercalcemic liabilityunlike 1,25(OH)₂D₃; and (3) it is easily synthesized.

For prevention and/or treatment purposes, the compounds of thisinvention defined by formula I and Ia may be formulated forpharmaceutical applications as a solution in innocuous solvents, or asan emulsion, suspension or dispersion in suitable solvents or carriers,or as pills, tablets or capsules, together with solid carriers,according to conventional methods known in the art. Any suchformulations may also contain other pharmaceutically-acceptable andnon-toxic excipients such as stabilizers, anti-oxidants, binders,coloring agents or emulsifying or taste-modifying agents.

The compounds of formula I and particularly 1-desoxy-2MD of formula Ia,may be administered orally, topically, parenterally, rectally, nasally,sublingually, or transdermally. The compound is advantageouslyadministered by injection or by intravenous infusion or suitable sterilesolutions, or in the form of liquid or solid doses via the alimentarycanal, or in the form of creams, ointments, patches, or similar vehiclessuitable for transdermal applications. A dose of from 0.01 μg to 1000 μgper day of the compounds I, particularly 1-desoxy-2MD, preferably fromabout 0.1 μg to about 500 μg per day, is appropriate for preventionand/or treatment purposes, such dose being adjusted according to thedisease to be treated, its severity and the response of the subject asis well understood in the art. Since the compound exhibits specificityof action, each may be suitably administered alone, or together withgraded doses of another active vitamin D compound—e.g. 1α-hydroxyvitaminD₂ or D₃, or 1α,25-dihydroxyvitamin D₃—in situations where differentdegrees of bone mineral mobilization and calcium transport stimulationis found to be advantageous.

Compositions for use in the above-mentioned treatments comprise aneffective amount of the compounds I, particularly 1-desoxy-2MD, asdefined by the above formula I and Ia as the active ingredient, and asuitable carrier. An effective amount of such compound for use inaccordance with this invention is from about 0.01 μg to about 1000 μgper gm of composition, preferably from about 0.1 μg to about 500 μg pergram of composition, and may be administered topically, transdermally,orally, rectally, nasally, sublingually or parenterally in dosages offrom about 0.01 μg/day to about 1000 μg /day, and preferably from about0.1 μg/day to about 500 μg/day.

The compounds I, particularly 1-desoxy-2MD, may be formulated as creams,lotions, ointments, topical patches, pills, capsules or tablets,suppositories, aerosols, or in liquid form as solutions, emulsions,dispersions, or suspensions in pharmaceutically innocuous and acceptablesolvent or oils, and such preparations may contain in addition otherpharmaceutically innocuous or beneficial components, such asstabilizers, antioxidants, emulsifiers, coloring agents, binders ortaste-modifying agents.

The compounds I, particularly 1-desoxy-2MD, may be advantageouslyadministered in amounts sufficient to effect the differentiation ofpromyelocytes to normal macrophages. Dosages as described above aresuitable, it being understood that the amounts given are to be adjustedin accordance with the severity of the disease, and the condition andresponse of the subject as is well understood in the art.

The formulations of the present invention comprise an active ingredientin association with a pharmaceutically acceptable carrier therefore andoptionally other therapeutic ingredients. The carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulations and not deleterious to the recipient thereof.

Formulations of the present invention suitable for oral administrationmay be in the form of discrete units as capsules, sachets, tablets orlozenges, each containing a predetermined amount of the activeingredient; in the form of a powder or granules; in the form of asolution or a suspension in an aqueous liquid or non-aqueous liquid; orin the form of an oil-in-water emulsion or a water-in-oil emulsion.

Formulations for rectal administration may be in the form of asuppository incorporating the active ingredient and carrier such ascocoa butter, or in the form of an enema.

Formulations suitable for parenteral administration convenientlycomprise a sterile oily or aqueous preparation of the active ingredientwhich is preferably isotonic with the blood of the recipient.

Formulations suitable for topical administration include liquid orsemi-liquid preparations such as liniments, lotions, applicants,oil-in-water or water-in-oil emulsions such as creams, ointments orpastes; or solutions or suspensions such as drops; or as sprays.

For nasal administration, inhalation of powder, self-propelling or sprayformulations, dispensed with a spray can, a nebulizer or an atomizer canbe used. The formulations, when dispensed, preferably have a particlesize in the range of 10 to 100μ.

The formulations may conveniently be presented in dosage unit form andmay be prepared by any of the methods well known in the art of pharmacy.By the term “dosage unit” is meant a unitary, i.e. a single dose whichis capable of being administered to a patient as a physically andchemically stable unit dose comprising either the active ingredient assuch or a mixture of it with solid or liquid pharmaceutical diluents orcarriers.

1. A compound having the formula:

where X is selected from the group consisting of hydrogen and ahydroxy-protecting group, and where R may be an alkyl, hydrogen,hydroxyalkyl or fluoroalkyl group, or R may represent a side chain ofthe formula:

where Z in the above side chain structure is selected from Y, —OY,—CH₂OY, —C≡CY and —CH═CHY, where the double bond in the side chain mayhave the cis or trans geometry, and where Y is selected from hydrogen,methyl, —COR⁵ and a radical of the structure:

where m and n, independently, represent the integers from 0 to 5, whereR¹ is selected from hydrogen, deuterium, hydroxy, protected hydroxy,fluoro, trifluoromethyl, and C₁₋₅-alkyl, which may be straight chain orbranched and, optionally, bear a hydroxy or protected-hydroxysubstituent, and where each of R⁵, R³, and R⁴, independently, isselected from deuterium, deuteroalkyl, hydrogen, fluoro, trifluoromethyland C₁₋₅ alkyl, which may be straight-chain or branched, and optionally,bear a hydroxy or protected-hydroxy substituent, and where R¹ and R²,taken together, represent an oxo group, or an alkylidene group having ageneral formula C_(k)H_(2k)— where k is an integer, the group ═CR²R³, orthe group —(CH₂)_(p)—, where p is an integer from 2 to 5, and where R³and R⁴, taken together, represent an oxo group, or the group—(CH₂)_(q)—, where q is an integer from 2 to 5, and where R⁵ representshydrogen, hydroxy, protected hydroxy, or C₁₋₅ alkyl and wherein any ofthe CH-groups at positions 20, 22, or 23 in the side chain may bereplaced by a nitrogen atom, or where any of the groups —CH(CH₃)—,—(CH₂)_(m)—, —CR₁R₂— or —(CH₂)_(n)— at positions 20, 22, and 23,respectively, may be replaced by an oxygen or sulfur atom.
 2. Thecompound of claim 1 wherein X is hydrogen.
 3. The compound of claim 1wherein R is selected from:


4. The compound of claim 3 wherein X is hydrogen.
 5. A pharmaceuticalcomposition containing an effective amount of at least one compound asclaimed in claim 1 together with a pharmaceutically acceptableexcipient.
 6. The pharmaceutical composition of claim 5 wherein saideffective amount comprises from about 0.01 μg to about 1000 μg per gramof composition.
 7. The pharmaceutical composition of claim 5 whereinsaid effective amount comprises from about 0.1 μg to about 500 μg pergram of composition.
 8. A compound having the formula:

where X₁ and X₂, which may be the same or different, are each selectedfrom hydrogen or a hydroxy-protecting group.
 9. The compound of claim 8wherein X₂ is hydrogen.
 10. The compound of claim 8 wherein X₁ ishydrogen.
 11. The compound of claim 8 wherein X₁ and X₂ are botht-butyldimethylsilyl.
 12. A pharmaceutical composition containing aneffective amount of at least one compound as claimed in claim 8 togetherwith a pharmaceutically acceptable excipient.
 13. The pharmaceuticalcomposition of claim 12 wherein said effective amount comprises fromabout 0.01 μg to about 1000 μg per gram of composition.
 14. Thepharmaceutical composition of claim 12 wherein said effective amountcomprise from about 0.1 μg to about 500 μg per gram of composition. 15.(20S)-25-hydroxy-1-desoxy-2-methylene-19-nor-vitamin D₃ having theformula:


16. A pharmaceutical composition containing an effective amount of(20S)-25-hydroxy-1-desoxy-2-methylene-19-nor-vitamin D₃ together with apharmaceutically acceptable excipient.
 17. The pharmaceuticalcomposition of claim 16 wherein said effective amount comprises fromabout 0.01 μg to about 1000 μg per gram of composition.
 18. Thepharmaceutical composition of claim 16 wherein said effective amountcomprises from about 0.1 μg to about 500 μg per gram of composition. 19.A method of treating a disease selected from the group consisting ofleukemia, colon cancer, breast cancer, skin cancer or prostate cancercomprising administering to a subject with said disease an effectiveamount of a 1-desoxy-2-methylene-19-nor-vitamin D analog having theformula:

where X is selected from the group consisting of hydrogen and ahydroxy-protecting group, and where R may be an alkyl, hydrogen,hydroxyalkyl or fluoroalkyl group, or R may represent a side chain ofthe formula:

where Z in the above side chain structure is selected from Y, —OY,—CH₂OY, —C≡CY and —CH═CHY, where the double bond in the side chain mayhave the cis or trans geometry, and where Y is selected from hydrogen,methyl, —COR⁵ and a radical of the structure:

where m and n, independently, represent the integers from 0 to 5, whereR¹ is selected from hydrogen, deuterium, hydroxy, protected hydroxy,fluoro, trifluoromethyl, and C₁₋₅-alkyl, which may be straight chain orbranched and, optionally, bear a hydroxy or protected-hydroxysubstituent, and where each of R⁵, R³, and R⁴, independently, isselected from deuterium, deuteroalkyl, hydrogen, fluoro, trifluoromethyland C₁₋₅ alkyl, which may be straight-chain or branched, and optionally,bear a hydroxy or protected-hydroxy substituent, and where R¹ and R²,taken together, represent an oxo group, or an alkylidene group having ageneral formula C_(k)H_(2k)— where k is an integer, the group ═CR²R³, orthe group —(CH₂)_(p)—, where p is an integer from 2 to 5, and where R³and R⁴, taken together, represent an oxo group, or the group—(CH₂)_(q)—, where q is an integer from 2 to 5, and where R⁵ representshydrogen, hydroxy, protected hydroxy, or C₁₋₅ alkyl and wherein any ofthe CH-groups at positions 20, 22, or 23 in the side chain may bereplaced by a nitrogen atom, or where any of the groups —CH(CH₃)—,—(CH₂)_(m)—, —CR₁R₂— or —(CH₂)_(n)— at positions 20, 22, and 23,respectively, may be replaced by an oxygen or sulfur atom.
 20. Themethod of claim 19 wherein the vitamin D analog is administered orally.21. The method of claim 19 wherein the vitamin D analog is administeredparenterally.
 22. The method of claim 19 wherein the vitamin D analog isadministered transdermally.
 23. The method of claim 19 wherein thecompound is administered rectally.
 24. The method of claim 19 whereinthe compound is administered nasally.
 25. The method of claim 19 whereinthe compound is administered sublingually.
 26. The method of claim 19wherein the vitamin D analog is administered in a dosage of from about0.01 μg/day to about 1000 μg/day.
 27. The method of claim 19 wherein thecompound is (20S)-25-hydroxy-1-desoxy-2-methylene-19-nor-vitamin D₃having the formula:


28. A compound having the formula:

where X is selected from the group consisting of hydrogen and ahydroxy-protecting group.
 29. The compound of claim 28 wherein X ishydrogen.
 30. The compound of claim 28 wherein X ist-butyldimethylsilyl.